Abstract 2350: Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer
Authors: Andrea Terrasi, Swathi Subramanian, Christine Klement, Sruthi Ramesh, Heike Bollig, Chiara Falcomatà, Katja Steiger, Rupert Öllinger, Dieter Saur, Roland Rad, Maximilian Reichert, Günter Schneider, Gunnar Schotta
Journal: Cancer Research (2022)

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer mortality within a decade with overall 5-year survival of 8% for all stages combined. Currently, it is well documented that mechanisms driving PDAC progression involve epigenetic and transcriptional rewiring. Here we combined assay for transposase-accessible chromatin using sequencing (ATAC-seq) and enrichment for H3K27 acetylation chromatin immunoprecipitation (H3K27ac ChIP-seq) measures to explore the epigenetic landscape of different mouse primary pancreatic tumor (PPT) cell lines. Methods: Kras-driven (n=36) and PI3K-driven PPT cell lines (n=9) were cultured in DMEM medium (Gibco). DNA was extracted using manufacture protocols (Qiagen, MinElute PCR Purification Kit) then DNA libraries and high-throughput sequencing were performed. Bioinformatics analysis (ROSE2 Python script) was conducted on H3k27ac ChIP-seq data to define super-enhancer (SEs) and SE-associated genes. Then, ATAC-seq data was explored using Coltron Python package to distinguish enriched Transcription Factor (TF) motifs into SEs. Transcriptomic data was used to slim down the list of potential cis-regulatory elements. We developed knockout (ko) PPT cell lines using CRISPR/CAS9 gene editing method to better characterize the role of Foxj1 as a novel potential master regulator in pancreatic cancer. Lastly, immunohistochemistry (IHC) staining for FOXJ1 was conducted on human PDAC cohort. Results: By k-means clustering, we identified 463 SE-associated genes. Many of them are associated with Kras-driven (epithelial or mesenchymal) or PI3K-driven cell lines exclusively. Surprisingly, we found Foxj1 as SE-associated TF exclusively in PI3K-driven PPT cell lines. Consistent with the epigenetic data, transcriptomic analysis confirmed higher expression of Foxj1 in PI3K-driven PPT cell lines. Then, RNA-seq data revealed downregulation of predicted Foxj1 target genes and enhanced EMT and Wnt/β-catenin signatures in Foxj1 ko cells. These data suggest that epithelial properties of PDAC cells are stabilized by Foxj1 activity. Consistent with these results we detect a higher potential of TGFβ treatment to induce mesenchymal features in Foxj1 ko cells. Furthermore, overexpression of β-catenin protein was confirmed by immunofluorescence. Enhanced Wnt/β-catenin signaling could be responsible for the higher proliferation of Foxj1 ko cells as revealed by proliferation assay. Finally, we investigated FOXJ1 protein level in our PDAC human cohort. Interestingly, we found high nuclear FOXJ1 expression in 23% of cases which is linked with better overall survival. Conclusions: In summary, our data revealed Foxj1 as a novel PDAC associated TF with the ability to reduce the cancer aggressiveness blocking epithelial to mesenchymal transition and β-catenin activity elucidating the better prognosis into the FOXJ1 high expressed patients. Citation Format: Andrea Terrasi, Swathi Subramanian, Christine Klement, Sruthi Ramesh, Heike Bollig, Chiara Falcomatà, Katja Steiger, Rupert Öllinger, Dieter Saur, Roland Rad, Maximilian Reichert, Günter Schneider, Gunnar Schotta. Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2350.